Most naturally occurring polypeptides contain carbohydrate moieties covalently attached to the polypeptide at some of the amino acids residues of the primary polypeptide chain. These polypeptides are generally referred to in the art as glycopeptides or glycoproteins. It is also known that the nature of the glycosylation pattern on any given polypeptide can affect its properties, including protease resistance, intracellular trafficking, secretion, tissue targeting, biological half life and antigenicity when the polypeptide is present in a biological system such as a cell or individual.
The glycosylation of polypeptides is a natural form of post-translational modification that alters the structure and function of polypeptides. In nature, glycosylation is introduced by enzymatic processes that lead to site specific modification of different types of glycosylated polypeptides. In N-linked glycosylation, glycans are attached to the amide nitrogen of asparagine side chains and in O-linked glycosylation, glycans are attached to the hydroxy oxygen of serine and threonine side chains. Other forms of glycosylation include glycosaminoglycans which are attached to the hydroxy oxygen of serine, glycolipids in which the glycans are attached to ceramide, hyaluronan which is unattached to either protein or lipid, and GPI anchors which link proteins to lipids through glycan linkages.
There is a general problem in the art in that glycosylation is often added to polypeptides in eukaryotic cells, but is rarely added to polypeptides expressed in the prokaryotic hosts often used for the recombinant expression of therapeutic polypeptides. This absence of glycosylation in polypeptides produced in prokaryotic hosts can lead to the polypeptides being recognised as foreign or mean that they have the properties that otherwise differ from their native forms. There is also a problem that it is difficult to engineer glycosylation into polypeptides at sites where there is not native glycosylation, in an attempt to use this to modulate the properties of the polypeptides.
There have therefore been attempts in the art to introduce or modify the glycosylation pattern of polypeptides, for example in situations where the expression of the polypeptide might cause a change to the natural glycosylation pattern of the polypeptide (e.g. expression in bacterial hosts) or where it is desired to modify the glycosylation pattern of the polypeptide in the hope of improving one or more of the characteristics of the polypeptide, especially where the polypeptide is a therapeutic protein. For example, see the modification of interferon beta described in U.S. Pat. No. 7,226,903.
The glycan molecules that are attached to polypeptides have a range of linear and branched structures and different lengths of glycan chain and the specific glycan molecules present on a polypeptide affects the characteristics of the polypeptide. Many types of glycan molecules include terminal sialic acids. These nine-carbon sugars, which bear a negative charge at physiological pH, are known to be involved in ligand-receptor interactions that can greatly affect specific cell-cell, pathogen-cell, or drug-cell communications. One particular characteristic of glycan chains that include terminal sialic acid residues is that they increase the half life of therapeutic polypeptides glycosylated with them. This is known from the observation that polypeptides comprising glycans without terminal sialic acid residues are rapidly removed from the circulation by the liver, thereby reducing the half-life of the therapeutic polypeptide.
Fluorine substituted sugars have been used as non processible substrates for use in crystallising enzymes such as CstII (Chiu et al., Nat. Struct. Mol. Biol. (2004) 11, 163-170). In this context, the fluorine containing saccharides are known to be resistant to enzymatic processing where the glycosyl transferase is acting upon the glycosidic linkage of the fluorine containing carbohydrate.
A 2,3-difluorosialic acid derivative has been synthesized and used as an inactivator of sialidases from the parasites Trypanosoma cruzi (Watts et al., J. Am. Chem. Soc. (2003) 125, 7532-7533) and Trypanosoma rangeli. (Watts et al., Can. J. Chem. (2004) 82, 1581-1588). This initial work led to the discovery that these sialidases operate through the involvement of a covalent sialosyl-enzyme intermediate and established that compounds such as this derivative acted as time-dependant covalent inactivators of Trypanosomal sialidases.
Subsequently, it has also been shown that a 2,3-difluoro neuraminic acid derivative which has a hydroxyl group at C-5 rather than the natural N-acetyl group also acts as a covalent inactivator of T. rangeli sialidase, but displays very different kinetic behaviour (kinact and kreact) to the original inhibitor (Watts et al., J. Biol. Chem. (2006) 281, 4149-4155).
In summary, the modification of the glycosylation of polypeptides, especially to modify their biological properties, remains a challenging problem and one that has not been addressed in a satisfactory manner in the prior art.